Miniaturized yeast identification system

ABSTRACT

A culture container having a convenient size for use under a conventional microscope stage. The container having media of a composition and amount effective to induce germ tube production in Cryptococcus. The media, which includes purified saponin, oxgall, a substrate for phenol oxidase, water and ammonia ions, is in an amount up to 5 milliliters at a depth up to 4.5 milliliters.

TECHNICAL FIELD

This invention relates to methods and compositions useful in the rapidand efficient identification of fungal pathogens. The methods andcompositions disclosed in the present invention utilize the differentialmorphologic and nutritive features of the genera and species ofpathogenic fungi sought to be identified. In one aspect of the presentinvention, fungal pathogens are differentially identified by methodsutilizing an improved formulation of a medium comprising a mixture ofpurified saponin, oxgall, a substrate for phenol oxidase, a supportingagent, and an ammonium salt. In another aspect, rapid and differentialidentification of fungal pathogens is provided by an improved method ofcarbohydrate assimilation, the improvement comprising preincubation ofsaid fungal pathogens in a carbohydrate depleted medium prior to theplating of said fungal pathogens on culture plates containing acarbohydrate assimilation medium. In another aspect, methods utilizingminiaturized culture plates, for example having the dimensions of33×75×5 millimeters, capable of differentiating fungal pathogens on fiveto fifteen fold less amount of fungal growth media than required bystandard culture plates, are described. In a further aspect of thepresent invention, methods employing surface inoculation procedures aredescribed which achieve a greater surface area to volume ratio thanconventional techniques. In yet another aspect of the present invention,methods utilizing a single miniaturized culture plate containing eithera combination of various fungal growth media or a number of differentfungal pathogens, or both, are described. In still a further aspect, theimproved fungal growth media, the improved carbohydrate assimilationmethod, and the methods utilizing miniaturized culture plates alone, andin combination, provide especially rapid and reliable techniques for thedetection and differentiation of three clinically important fungi:Candida albicans, Candida stellatoidea and Cryptococcus neoformans.

BACKGROUND ART

Fungal pathogens vary in their pathogenicity, that is, their capacity tocause disease. At one end of the spectrum, highly pathogenic fungi mayproduce systemic disease when they infect. At the other end of thespectrum, some fungi may establish a more or less permanent residenceon, and even in, the superficial body tissue. These fungi are unable topenetrate the body's natural defenses unless these defenses areseriously impaired. Such opportunistic infections have become morecommon as a byproduct of therapeutic advances and currently pose asignificant medical threat to certain classes of individuals orpatients. For example, patients receiving antibiotic therapy, undergoingsurgical procedures, manipulation of indwelling catheters and those withaltered host defense mechanisms due to the use of antibiotics,immunosuppressive drugs or radiation therapy have an increased risk ofopportunistic fungal infection. Fungal infections in these alreadycompromised individuals may result in life threatening disease.

Two of the most medically important types of fungi are Candida albicansand Cryptococcus neoformans. Candida albicans is an example of arelatively non-pathogenic yeast (that is, single cell fungus) that isfrequently present on normal mucus membranes of the mouth and intestinaltract. Candida albicans generally will not establish an infection inhealthy humans but can cause opportunistic infections resulting inchronic local infections in those with compromised host defensemechanisms. Cryptococcus neoformans is especially important because ofits predilection for the central nervous system which can cause severedisease in biologically defenseless patients.

The presumptive identification of Candida albicans depends solely uponmorphological changes which occur when this fungus is plated and allowedto grow on an appropriate medium. The first morphological changeindicative of the presence of Candida albicans is the formation of germtubes, which appear as tiny appendages extending from the platedunicellular specimens. These germ tubes eventually grow into elongatedfilaments extending outwardly from a body of the Candida albicans.Formation of germ tubes within 2 to 3 hours after plating of the fungusis presumptive evidence that Candida albicans or Candida stellatoideaare present. In a second stage of growth, generally round bodies appearat the ends of the filaments. These round bodies are known aschlamydospores. Only three species of the Candida genus will formchlamydospores. These are Candida albicans, Candida stellatoidea, andCandida tropicalis. Thus, chlamydospore formation is indicative of thepresence of Candida albicans, Candida stellatoidea, or Candidatropicalis.

The differential identification of Candida albicans and Candidastellatoidea is made by a subsequent carbohydrate assimilation test. Thebasic concept for these tests includes a series of differential mediawhich contain one specific carbohydrate, a nitrogen source and a colordye such as bromcresol purple which is pH sensitive. For yeasts,carbohydrate assimilation is an acidic reaction which can be monitoredwith pH-indicating dyes, while fermentation involves acid and gasproduction, requiring a pH-indicating dye and gas trap.

There are two methods presently used for assimilation: (1) a modifiedWickerham method and (2) an auxanographic method. In general, themodified Wickerham method uses test tubes of carbohydrate agar mediawhich contain a pH sensitive dye. The tubes are inoculated directly froma yeast subculture plated on a growth medium such as Sabouraud'sdextrose agar by transferring a loop of organism to the surface of thecarbohydrate agar. Positive assimilation reactions are noticeable in oneto seven days. A conventional example of this method includes theinoculation of a test tube of sucrose media containing bromcresol purplewith yeast followed by an examination at 24 to 72 hours for a colorchange of the medium from purple to yellow indicating sucroseassimilation. Candida albicans will give a positive assimilation whileCandida stellatoidea is negative. The basic disadvantages are a longtime-to-positivity and cumbersome and large storage space requiringspecialized racks for storage.

Another conventional plate also utilizes the modified Wickerham methodwith one basic alteration. The plate is composed of a series of wedgesarranged in a circular configuration which contain a carbohydrate mediawith bromcresol purple, an urea agar and a nitrate media. Instead of adirect inoculum transfer, the yeast is first suspended in sterile waterand then transferred by sterile pipet to the various media. The plate isthen incubated and examined at 24 hours to 6 days for positivereactions. Disadvantages of this system are long time-to-positivity,relative expense and an increased amount of media used, each wellrequiring about ten milliliters of media.

The auxanographic method involves a pour plate technique where the yeastis suspended into molten carbohydrate agar medium. The yeast suspensionis then poured into a sterile culture (hereinafter sometimes referred toas "petri") plate and allowed to solidify. There are two variations ofthis method. In one method, the medium does not contain carbohydrate,and after solidification, sterile paper discs impregnated with aspecific carbohydrate are placed onto the agar surface of the inoculatedpour plate. The carbohydrate then diffuses from the disc into theinoculated medium. Positive assimilation is then measured either by a pHchange around the disc in the presence of a pH sensitive dye or by aring of turbidity forming around the paper disc indicating the abilityof the yeast to assimilate, or to grow on, that particular carbohydratesource. Sterile carbohydrate discs are available which can beincorporated into an identification system. With this system, eight ortwelve different carbohydrate discs can be delivered in one applicationto a standard 100×15 mm petri plate of inoculated yeast assimilationmedium. The disadvantages of this method are the necessity of a boilingbath to melt the agar medium, prolonged technician time, cumbersomestorage, and the long time-to-positivity. Additionally, the temperatureof the molten agar is typically between 40° and 45° C. at the time ofyeast suspension in said agar. Temperatures above these temperatures cankill yeast cultures and thus produce false negatives in the assimilationtest results.

Another approach to the auxanographic method is illustrated by theA.P.I. 20C System. In this system, a series of twenty wells containinglyophilized carbohydrates and a glass vial filled with the basic yeastassimilation medium, without a pH sensitive dye, is provided. The glassvial is placed in a boiling water bath to melt the agar medium. Aftercooling, the yeast is transferred to the medium using a sterile woodenapplicator stick and the medium is stirred to make a uniform suspension.This suspension, is then transferred by sterile pipet to the nineteencarbohydrate wells and one negative growth control well and allowed tosolidify. The inoculated strip of wells is then placed in a plasticincubation tray provided by A.P.I. and incubated at 30° C. This systemrequires reactions to be read at 24, 48 and 72 hours with a positivereaction determined by increasing turbidity. Disadvantages of thissystem are the necessity of a boiling water bath to melt the agarmedium, technician time, long time-to-positivity, and relative expense.Additionally, the temperature of the melted agar is typically between40° and 45° C. at the time of yeast suspension in said agar.Temperatures above these temperatures can kill yeast cultures and thusproduce false negatives in the test results.

Identification of Cryptococcus neoformans is generally recognized to bemore difficult than the identification of Candida albicans in thatCryptococcus neoformans undergoes no morphological changes which can beobserved and remains unicellular throughout its growth cycle. Untilrecently, one or more of three basic tests, or a combination thereof,were employed to identify the presence of the genus Cryptococcus. One ofthe methods of identification comprises microscopic inspection of aspecimen to identify whether or not a capsule-like formation around thecells of the fungi is present. In order to aid in the inspection of suchcapsule-like formations, a specimen is surrounded with india ink whichenhances the appearance of the capsule by providing a clear andtranslucent image against the black background making such capsuleseasier to identify during microscopic examination. A second methodemployed to identify the genus Cryptococcus comprises plating thespecimen on a medium containing urea and a color indicator. BecauseCryptococcus produces an enzyme known as urease it has the capability tobreak down and use the nitrogen contained in the urea, causing the pH torise, thereby changing the color of the indicator. Therefore, metabolismof a urea containing medium is indicative of the presence ofCryptococcus, Trichosporon beigelii or Candida krusei. A third method ofidentification of the genus Cryptococcus relies on the ability of thatgenus to produce a starch-like compound. When the starch-like compoundis present, addition of iodine will cause a purple ring to appear aroundthe colony. It is to be noted that none of these tests is specific forCryptococcus neoformans by itself or in combination, as other specieswithin the genus Cryptococcus and other genera of yeast may also give apositive reaction.

Although the urease test, described above, is not specific forCryptococcus neoformans, it is a relatively rapid preliminary screen orthe genus Cryptococcus which characteristically possess the enzymeurease that is necessary for the hydrolysis of urea. Three methods arecurrently employed to detect urease production by yeast cultures: (1)urea agar slant, (2) urea broth and (3) urease swab test. The urea agarslants are prepared from commercially dehydrated medium containing acolor indicator. After autoclaving, slanting and solidification, thetubes are ready to use. Positive urease reactions are noted by a colorchange of the medium from orange to pink which occurs within 24 to 72hours. The primary disadvantages are long time-to-positivity andtechnician time.

The urea broth test essentially changes the test to a liquid state andincreases the inoculum to substrate ratio. The urea broth can bepurchased commercially in a lyophilized form and must therefore bereconstituted prior to use. This test requires 3 to 4 hours for positivereactions incicated by a color change of the broth from orange to pink.Tne major disadvantages are preparation time and desirability of an evenmore rapid preliminary screen.

The urea swabs are prepared by impregnating sterile cotton applicatorswabs with concentrated urea agar base, quick-freezing at -70° C. andlyophilizing overnight. The swabs are inoculated with 2 to 3 yeastcolonies, placed into a test medium, the pH of the media is adjusted topH 4.6, then incubated and examined for a positive reaction indicated bya color change which occurs within 15 to 20 minutes. The basic problemwith this test is that the swabs are not commercially available and thepH adjustment for the swab is so critical that there is a likelihood offalse negative or false positive reactions occuring.

Perhaps the single most successful and specific conventional test forCryptococcus neoformans includes the use of bird seed agar. It wasdiscovered that when Cryptococcus neoformans was present in a sampleplated on bird seed agar a specific tell-tale brown color would appear,within a period of five days to two weeks. Tnis method was improved byusing an extract of bird seed which lowered the identification time 3 to5 days. Later it was discovered that the brown pigment coloration of theyeast was the result of the reaction between the enzyme phenol oxidaseand a particular substrate present in bird seed agar. Accordingly, useof substituted phenols such as caffeic acid in the growth medium furthershortened the period of time necessary for identification to aboutforty-eight hours. A still further refinement of the use of caffeic acidto identify Cryptococcus neoformans is set forth in an article by Hopferand Groschel entitled "Six Hour Pigmentation Tests for theIdentification of Cryptococcus neoformans", Journal of ClinicalMicrobiology, August 1975, Vol. II, No. 2, p. 96-98. The improvement setforth therein includes combining caffeic acid with ferric citrate andincorporating these compounds onto paper discs for use as substrates forthe phenol oxidase enzyme activity of Cryptococcus neoformans. Use ofthese caffeic acid-ferric citrate impregnated paper discs further lowersthe identification time to 3 to 6 hours. However, the solution ofcaffeic acid and ferric citrate used to impregnate the paper discs isquite unstable when exposed to light and temperature and thereforepresents serious storage problems. These discs must be stored at -20° C.Furthermore, the relative concentration of caffeic acid and ferriccitrate are critical and an unbalanced combination will require longerincubation periods for production of a dark pigment, or, in some cases,nonspecific pigmentation of saprophytic Cryptococcus and several Candidaspecies. In an article entitled "Two Rapid Pigmentation Tests forIdentification of Cryptococcus neoformans," Journal of ClinicalMicrobiology, February, 1982, Vol. 15, No. 2, p. 339-341, by Kaufmannand Merz, the authors present two tests for Cryptococcus neoformansbased in part upon modifications of previous approaches to detect phenoloxidase activity. The first test employs corn meal agar containing anemulsifying agent sold under the tradename Tween 80 by Atlas ChemicalCompany supplemented with caffeic acid. The second is a non-medium testutilizing a phenol oxidase detection strip saturated with bufferedL-β-3,4-dihydroxyphenylalanine (L-DOPA)-ferric citrate solution. Whilesubstrate stability is increased in these tests, storage still presentssome problems as ferric citrate, for example, must be stored at -20° C.

Recently, a new culture medium for the identification of Cryptococcusneoformans, Candida albicans and Candida stellatoidea was discoveredwhich includes caffeic acid, oxgall, saponin and a supporting agent.This medium (hereinafter sometimes referred to as original SOC) isdisclosed in U.S. Pat. No. 4,144,133, issued Mar. 13, 1979 and entitled"Fungal Growth Media". The phenol oxidase substrate, preferably in theform of caffeic acid, provides for a specific identification ofCryptococcus neoformans by means of the appearance of the characteristicbrown pigmentation of the yeast which results from specific enzymeactivity of Cryptococcus neoformans on the phenol oxidase substrate. Theoxgall, in addition to its known function of suppressing bacterialgrowth, has also been discovered to enhance filament and chlamydosporeproduction of the medically important fungi Candida albicans and Candidastellatoidea. The purified saponin employed in the fungal mediasignificantly enhances the germ tube and chlamydospore formation ofCandida albicans thus providing for rapid identification of theseespecially serious types of pathogenic fungi. The carrying agent, suchas common agar or silica gels for example, simply provides a supportingbase for the above described active ingredients.

Although the SOC culture media are specific for identification ofCryptococcus neoformans and rapidly indicate the presence of either thealbicans or stellatoidea species of the genus Candida, chlamydosporeformation is weak in some strains of Candida albicans requiring 48 to 72hours for formation rather than 28 to 48 hours for the strongchlamydospore formers.

Thus, while a variety of methods and media have been employed in orderto identify and differentiate various fungal pathogens including thecritically important genera and species Candida albicans andCryptococcus neoformans, there is a continuing need for a fungal growthmedia and system which will rapidly identify and differentiate thesegenera and species as well as other pathogenic fungi in a rapid,efficacious, economic and technically efficient manner.

SUMMARY OF THE INVENTION

The present invention provides a miniaturized yeast identificationsystem which allows for the rapid and reliable identification of themost commonly encountered opportunistic fungi infecting patients withcompromised body defenses. Existing and improved differential fungalgrowth media are modified or adapted to miniature culture containershaving dimensions such that it will fit on the viewing stand of aconventional microscope, for example a size of approximately 33×75×5millimeters. Preferably flip-up lid is pivotally attached to thesidewalls which when in the lowered or closed position will enclose themedia within the container, but when pivotally open will allow easyaddition, removal and inspection of the samples. These miniature plateshave a physical form which is convenient for storage, examination andeasily maintained sterility. The miniature configuration also affectsthe performance of various differential growth media. Specifically, thelarger surface area to volume in the miniature plate allows rapid heattransfer between the incubation environment and the medium thusproviding an enhanced temperature control essential with the SOC medium,and provides an easily attainable high organism to substrate ratiorequired by the carbohydrate assimilation and urea containing media, forexample, from about 10⁵ to about 10¹² organisms per about 0.2centimeters squared to about 5 centimeters squared media. The presentinvention additionally provides an improved SOC medium, the improvementcomprising the addition of ammonium ions. The addition of ammonium ionsenhances chlamydospore formations in previous-y weak chlamydosporeforming strains of Candida albicans. Additionally, this improved SOCmedium has been further modified by increasing the concentration of thephenol oxidase substrate, caffeic acid, to allow for the adaptation ofthe SOC medium to the miniaturized system. The methods of the presentinvention further describe the use or these miniature plates so as toaccommodate one or more organisms, one or more differential growthmedia, or a combination thereof to provide a compact and rapid yeastidentification system.

BRIEF DESCRIPTION OF THE DRAWINGS

One aspect of the present invention, the novel miniature culturecontainer, has dimensions such that it will fit on the viewing stand ofa conventional microscope, for example, a size of approximately 33×75×5millimeters. The several embodiments of the novel containers can be moreeasily understood from a study of the drawings in which:

FIGS. 1-4 are perspective views of four distinct embodiments of thenovel miniature culture container.

FIG. 5 is an enlarged fragmentary cross-sectional view of the structureof FIG. 1.

FIGS. 6-11 are perspective views of six distinct embodiments of thenovel miniature culture container.

FIG. 12 is an enlarged fragmentary cross-sectional view of FIG. 10 takenalong lines 12--12.

FIG. 13 is a graph showing the effect of fungal medium depth on thetime-to-positivity in the carbohydrate assimilation test of Example 7.

DETAILED DESCRIPTION

The present invention relates to methods and compositions useful in therapid and efficient identification of fungal pathogens.

In the context of this disclosure, the following terms shall be definedas follows unless otherwise stated:

"blastospore" is a spore formed by budding as in yeast (syn: yeast,yeast cell);

"chlamydospore" is a thick-walled, non-deciduous asexual spore made bythe rounding up of a cell either intercalary (at the junction of twocells in a filament) or terminally (at the end of a filament);

"filament" is a thread-like series of elongated yeast cells;

"germ tube" is a hyphal extension from a yeast cell which has noconstriction or cell wall at the point of production;

"pseudohypha" is a hyphal extension from a yeast cell formed by theelongation of a budding cell where constriction and cell walls separateone yeast cell from the other;

"pseudomycelium" is a mass of filaments formed by elongated yeast cells;

"yeast" is a unicellular, budding fungus.

The miniaturized yeast identification system of the present inventionprovides an especially rapid, reliable, economic and technicallyefficient method for the differentiation of most medically importanttypes of potentially pathogenic fungi.

The yeast identification system contains at least one differentialmedium which is provided in a miniature culture plate described in moredetail below. The media include an improved fungal growth medium, theimprovement comprising the addition of ammonium ions to a mediumcomprising purified saponin, oxgall, a substrate for phenol oxidase anda supporting agent (hereinafter sometimes referred to as SOC),carbohydrate assimilation media and a urease medium. The miniature mediacontaining plates may be constructed to contain a single medium, asshown in FIGS. 1 through 4, or may be partitioned, as shown by examplein FIGS. 6 through 11. It is envisioned that plates may be sopartitioned to accommodate as many as twenty different media.

Generally, saponins may be purified using the techniques set forth inU.S. Pat. No 3,883,425 entitled "DETOXIFICATION OF SAPONINS", issued May13, 1975, incorporated by reference herein, and the purified saponinscan then be employed in the fungal medium of the present invention. Theuse and adaptation of these media to miniature culture platessignificantly enhances the performance of these media in differentiallyidentifying various genera and species of clinically important fungi.The miniaturized yeast identification system of the present inventionalso includes a modification of the carbohydrate assimilation test, saidmodification comprising the preincubation of the fungi in a carbohydratedepleted medium prior to plating on a carbohydrate assimilation media.The described modification significantly decreases thetime-to-positivity, specifically, from 24 to between four and six hoursfor some species. The use and adaptation of these media to theminiaturized system provides both a surface inoculation method andgreater surface area to volume ratio than conventional methods which canreduce the number of false negative identifications that may occur whenpour plate methods are used. This miniaturization also has economic andmechanical advantages. The miniaturized yeast identification system ofthe present invention is particularly useful in the differentialidentification Cryptococcus neoformans, Candida albicans and Candidastellatoidea.

The miniature plate has dimensions such that it will fit on the viewingstand of a conventional microscope, for example of from about 0.03 toabout 3 centimeters by from about 0.7 to about 8 centimeters with adepth of from about 0.025 centimeters to about 1.0 centimeters. Thepreferred size is approximately 35×75×5 millimeters, embodiments 10 and20 in FIGS. 1 and 2 respectively, and has a flip-up lid 12 which ispivotally attached to the sidewalls 14a and 14b and which when in thelowered position, see FIG. 5, will enclose the media 16 within the plate18 but when pivotally open, see FIG. 1, will allow easy addition,removal and inspection of the samples. The standard petri plate is acircular plate with the approximate dimensions of 100×15 millimeters andhas a non-attached lid which fits over the bottom, media containing,plate. The miniaturized configuration has several advantages.Specifically, these advantages include ease of packaging and storage,requiring little refrigeration room, and increased facility inmaintaining sterility as the plates are easily handled and stackedwithout the fear of knocking off the lid which is a common occurrencewith conventional petri plates. Additionally, with the morphology mediumSOC, the miniature plate configuration fits the microscope stage slideholder allowing rapid manipulation of the plates as opposed to the slow,awkward manipulation of standard petri plates by hand and by eliminatingthe need to transfer the yeast specimen to a second microscope slide forexamination.

Referring to the drawings, FIGS. 1, 2, 8, 9, 10 and 11 representrectangular embodiments of the miniature culture plate of the presentinvention. As shown by example in FIGS. 1 and 2, these embodiments maypossess a flip-up lid 12 which is pivotally attached to side walls 14aand 14b may possess an unattached lid 22. Both lids 12 and 22 may belowered upon plates 18 and 28, respestively, to enclose the media 16contained within plates 18, 28, 50, 54, 60 and 70 in a manner best shownin FIG. 5. As shown by example in FIGS. 8 through 11, the rectangularplates 18 and 28 of embodiments 10 and 20, respectively, may bepartitioned in such a manner as to create circular wells 62a, 62b,72a-h, shown in detail in FIG. 12, square wells 55a-h, or rectangularwells 52a-c, and 64a-b or a combination thereof as shown in embodiment60. Alternatively, the miniature culture plates may be circular inconfiguration as shown by embodiments 30 and 40 in FIGS. 3 and 4,respectively. The circular plates 38, 48, 80 and 90 may similarlypossess a flip-up lid 32 which is pivotally attached to thecircumferential sidewall 34 or may possess an unattached lid 42, both ofwhich enclose the media 16 contained within plates 38, 48, 80, and 90when the lids 32 and 42 are lowered upon the circumferential sidewalls34 and 44, respectively. In a manner similar to the rectangular plates18, 28, 50, 54, 60 and 70, the circular plates 38, 48, 80 and 90 may bepartitioned as shown in FIGS. 6 and 7 to create wells of such shapes ascircles 92a and 92b or triangular pie-shaped wells 82a-f and 94a-d orany combination thereof, for example embodiment 90, FIG. 7. The shape ofthe miniature culture plate and media containing wells therein, shown inFIGS. 1 through 12. are set forth as examples and are not meant to limitthe types of possible shapes and combinations thereof employed. Theminiature culture plate may be constructed of plastic or such materialpresently employed in the manufacture of petri plates.

The miniature plates are more economical in that they utilize betweenabout 2.5 to 5 milliliters of media as opposed to the about 20 to 30milliliters of media required by the standard petri plates. Theminiaturized yeast identification system has been designed to utilize aslide warmer as an alternative mini-incubator. One such slide warmer iscommercially available from Fisher Scientific Company. There are tworequirements for use of slide warmers: (1) an adjustable thermostatwhich can be set at 37° C. and (2) a cover or lid for the warmingsurface. Both the purchase cost and space needs of a slide warmer areconsiderably less than a conventional incubator.

The miniaturized yeast identification system of the present inventionprovides pre-poured, sterilized, ready to use media and therebycircumvents the disadvantages of existing systems, such as theauxanographic carbohydrate assimilation method which necessitates suchsteps as boiling, to dissolve the agar supporting agent andsterilization prior to use. A conventional product with a ready-to-useagar for chlamydospore production uses corn meal agar with anemulsifying agent sold under the tradename Tween 80 by Atlas ChemicalCompany and accompanies media for urea, nitrate and carbohydrateutilization. The corn meal agar is inoculated and examined at 24 to 72hours for chlamydospore production. The two main disadvantages of thissystem are that the chlamydospore tests cannot be performed apart fromutilizing the entire plate which is relatively expensive and there is along time-to-positivity.

In the miniaturized yeast identification system of the presentinvention, the media are prepared, poured into the miniature plates andafter solidification, the lids are closed, and the plates are wrapped ina plastic lined aluminum pouch. These plates may be stored up to atleast 8 months at 4° C. without loss of differential growthcapabilities.

The miniature configuration also affects the performance of the media.Temperature control is essential with these media and the smaller volumeof media in the miniature plate allows rapid heat transfer betweenincubation environment and the medium. The sensitivity and accuracy ofshort term incubations, such as the three hour, 37° C. incubation forgerm tube formation and subsequent shift to room temperature forchlamydospore production on the SOC medium, are significantly enhancedby the miniaturization of the system. The performance of the sucroseassimilation and urease tests is also enhanced. Both of the latter testsrequire a very high organism to substrate ratio which is easilyattainable in the small media volume of the miniature plate.Additionally, both tests involve a color change of the media which ismore easily visualized with a medium depth of about 1 to 4.5 millimetershan at about 5 to 15 millimeters. This increased organism to substrateratio and more easily visualized color change are thought to bepartially responsible for the decreased time-to-positivity of thesucrose and urease tests in the miniaturized system of the presentinvention, the times being 5 and 1 hours, respectively.

Use of the SOC media in the miniaturized system of the present inventionrequires a modification in the concentration of the phenol oxidasesubstrate. The preferred substrate is caffeic acid, however, some othersuitable substrate of phenol oxidase enzymes may be employed. Suitablepnenol oxidase substrates include 2,3-dihydroxybenzoic acid,3,4dihydroxybenzoic acid (protocatechuic acid), DOPA,3,4-dihydroxycinnamic acid (caffeic acid), the methyl ester anddiacetate of caffeic acid, 3-hydroxytryptamine,3,4-dihydroxyphenylethanolamine (norepinephrine), and4-hydroxy-3,5-dimethoxycinnamic acid. The substrate of phenol oxidase isemployed as an identification agent for Cryptococcus neoformans sincethe reaction of the phenol oxidase enzyme of that fungus with such asubstrate produces a brownish pigmentation of the organism which isspecific for Cryptococcus neoformans. The original SOC formulation, asgiven in U.S. Pat. No. 4,144,133, issued Mar. 13, 1979 and entitled"FUNGAL GROWTH MEDIA", was from about 0.005 to about 0.05 weight percentcaffeic acid. Adaptation of the original SOC media to the miniaturizedsystem requires an increased range of about 0.005 to about 0.5 weightpercent, with a preferred concentration of about 0.012 to about 0.12weight percent and a most preferred concentration of about 0.06 weightpercent caffeic acid. This higher concentration of caffeic acidnecessitates an upward adjustment of the pH of the medium to a neutralpH due to the acidic nature of caffeic acid.

It was noted that chlamydospore formation, in certain strains of Candidaalbicans, was very weak on the original SOC media, requiring a 48 to 72hour incubation for production. Publications by McClary, Annals MissouriBot. Gar., 39:137-164 (1952) and Nickerson, Internat'l. Congress ofMicrobiol. Report Proceedings, 5th Congress, Rio de Janeiro, p. 130-131(1950), indicated that low concentrations of ammonium sulfate orammonium chloride support filamentation in Candida species. Other workby Jansons, et al., J. Bacteriol, 104, 2:910-921 (1970); Land, et al.,Infect. and Immun., 11, 5:1014-1023 (1975); Mardon, et al., J.Bacteriol, 100:701-707 (1969); and Nickerson, ibid, stated that ammoniumsalts support yeast growth. Twenty strains of Candida albicans werechosen, which on occasion had given negative or weak chlamydosporeformation, and were tested for germ tube and chlamydospore formation onSOC media containing a range of ammonium ion concentrations in theminiaturized system. All twenty strains produced numerous germ tubes onammonium ion containing SOC media after three hours at 37° C. Theconcentration range of ammonium ions, added in the form of ammoniumsalts, supporting chlamydospore formation was found to be between about0.001 M and about 0.5 M with a preferred concentration of about 0.005 Mto about 0.05 M and a most preferred concentration of ammonium salt atabout 0.01 M. Ammonium salts may be selected from a non-exclusive classcomprising, ammonium chloride, ammonium sulfate, ammonium nitrate andammonium citrate, of which the preferred ammonium salt is ammoniumchloride. Other salts such as sodium chloride and potassium chloridewere tested by formulation in the SOC media but were found to be lesseffective in promoting chlamydospore formation than the ammonium salts.

Miniaturization of the SOC medium also required an increase in thepreferred concentration of purified saponin from the originalconcentration of about 0.5 weight percent to about 1.0 weight percent inthe improved SOC medium of the present invention.

The SOC medium used in the miniaturized yeast identification system wasprepared by adding a powdered supporting agent such as agar, oxgall,purified saponin, a substance for phenor oxidase such as caffeic acid,and ammonium salt to deionized water. The resulting mixture was thenstirred and heated to boiling to dissolve the ingredients and adjustedto a final pH of about 6.5 to about 7.5. The solution was thensterilized at 121° C. at about 15 psi for 15 minutes. After cooling, themedium was dispensed to a depth of about 2 millimeters into 33×75×5 mmrectangular plastic plates with attached flip-up lids. The medium wasallowed to solidify, and the plates were wrapped in plastic linedaluminum foil pouches and stored at 4° C. Preparation and storage inthis fashion gives a shelf life of at least 8 months.

Generally, the improved SOC medium of the present invention comprisesfrom about 1.0 to about 5.0 weight percent agar, from about 0.25 toabout 30.0 weight percent oxgall, from about 0.1 to about 5.0 weightpercent purified saponin, from about 0.005 to about 0.5 weight percentcaffeic acid, based on the amount of water added to these components andfrom about 0.001 M to about 0.5 M ammonium salts.

Preferred media can be prepared following the procedure outlined aboveand employing from about 1.0 to about 5.0 weight percent agar, fromabout 0.5 to about 5.0 weight percent oxgall, from about 0.1 to about2.5 weight percent purified saponin, from about 0.012 to about 0.12weight percent caffeic acid, and from about 0.005 M to about 0.05 Mammonium salts. A most preferred medium contains about 2.0 weightpercent agar, about 1.0 weight percent oxgall, about 1.0 weight percentpurified saponin, about 0.06 weight percent caffeic acid, and about 0.01M ammonium salts.

Prior to inoculation of the miniature SOC media-containing plates withthe yeast samples, the yeasts are grown on a general growth medium, suchas Sabouraud dextrose agar with gentamicin, for up to about 72 hours.Anywhere from about one to three yeast colonies may be taken from thegeneral growth medium and plated on each miniature plate. All yeastswere first grown on a general growth media prior to inoculation ofmedia-containing miniature plates. These general growth media maycontain antibiotics.

The miniaturized yeast identification system of the present inventionhas also been adapted to accommodate carbohydrate assimilation media.Adaptation of these assimilation media to the miniaturized system allowsclinical technicians to selectively choose those assimilations requiredfor a specific speciation of potential pathogenic fungi and therebyshortens the time of species identification from one week to about fiveto twenty four hours. Sucrose assimilation, for example, specificallydifferentiates Candida albicans, which assimilates sucrose, from Candidastellatoidea, which does not. When used in conjunction with suchmorphology tests as germ tube and chlamydospore formation, which gives apresumptive identification of the albicans or stellatoidea species, theconducting of sucrose assimilation tests will give a conclusive speciesidentification. Additionally, adaptation of such carbohydrateassimilation media as sucrose has dramatically shortened thetime-to-positivity for Candida albicans and Candida stellatoideadifferentiation.

The method of the present invention shortens the time-to-positivity forsucrose assimilation from 24 hours, and in some tests 6 days, to aperiod of about 5 hours. This increased efficiency is believed to be dueto at least two novel features of the present invention. First, theprocedure employed for sucrose assimilation utilizes a starvation step,in a carbohydrate depleted media, prior to plating of the fungi on theassimilation media. The purpose of this prestarvation step is to depletethe yeast cells of essentially all of the internal carbohydrate poolswhich prevents false positive reactions. This prestarvation is believedto increase the rate of carbohydrate assimilation once the yeasts areplated on carbohydrate containing media and thus decrease thetime-to-positivity. Second, the miniature plates have a media depth ofabout 1 millimeter to about 4.5 millimeters as opposed to 5 millimetersto 15 millimeters in the standard petri plate. The shallower depth ofthe miniature plates is thought to aid in a more rapid visualization ofthe color change resulting from positive carbohydrate assimilation andto provide the essential high organism to substrate ratio.

In the miniaturized yeast assimilation system, a sucrose assimilation isutilized to differentiate between Candida albicans and Candidastellatoidea. The medium was prepared by adding a powdered supportingagent, such as agar, a pH sensitive color dye, such as bromcresolpurple, and a yeast nitrogen base to deionized water. The resultingmixture is then stirred, heated to boiling to dissolve the mediacomponents and adjusted to a pH of about 6.85 to about 7.55 with a base,such as sodium hydroxide. The medium is then sterilized by heating to121° C. at 15 psi for 15 minutes. The sucrose is presterilized and thenadded asceptically to the medium mixture. The cooled sucrose-containingmedium is then dispensed in about 5 milliliter aliquots into sterilerectangular plates with attached flip-up lids, the plates having theapproximate dimensions of 33×75×5 millimeters. After solidification, thelids are closed, and the plates wrapped in plastic lined aluminum foilpouches and stored at 4° C. These plates may be stored up to at least 8months at 4° C. without dehydration or loss of color. Other sugars, suchas maltose, galactose, trehalose, and lactose, may be substituted forsucrose to provide additional carbohydrate assimilation media. Thesesugars are but non-exclusive examples of carbohydrates which may besubstituted for sucrose in the above-described carbohydrate assimilationmedia. Additionally, it is envisioned that a series of differentcarbohydrate assimilations may be conducted simultaneously by utilizingcontainers such as those shown in FIGS. 6 through 11. Fluorescentindicators which show a definite change in fluorescence with change inpH may be substituted for the pH sensitive color dyes employed in theexamples of the present invention. Fluorescent indicators operable inthe pH range, pH 5 to pH 8, of the carbohydrate assimilation systeminclude, for example, Acid R Phosphine, Brilliant Diazol Yellow, Clevesacid, Coumaric acid, 3,6-Dioxyphthalic dinitrile, Magnesium8-hydroxyquinolinate, β-Methylumbelliferone, 1-Naphthol-4-sulfonic acid,Orcinaurine, Patent Phosphine, Thioflavine and Umbelliferone.

A preferred medium can be prepared following the procedure outlinedabove and employing from about one to about five weight percent agar,from about 0.0005 to about 0.02 weight percent bromcresol purple, fromabout 0.02 to about 0.7 weight percent yeast nitrogen base, and about0.02 to about 1.0 percent sucrose, based on the amount of water added tothese components. The preferred pH of the medium, prior to sterilizationis about 6.85 to about 7.55.

Prior to inoculation of the assimilation media, the yeasts were takenfrom plates containing a general growth medium and suspended in sterile,carbohydrate depleted media at a pH of about 7.0 to about 8.5 and heldat room temperature. Examples of carbohydrate depleted media includesterile, deionized water or deionized water supplemented with a yeastnitrogen base. The prestarvation period may run from about 30 minutes to24 hours with a preferred range from about 30 minutes to about 8 hours,and a most preferred period of about one hour. Prestarvations werecarried out in non-glass, sealed and sterile containers. Suchprestarvation techniques have been employed in genetic and biochemicalstudies of microorganisms generally, but have not been previouslyemployed in yeast assimilation methods for taxonomic purposes.

After prestarvation, the carbohydrate depleted medium containing theyeast is vigorously mixed and, thereafter, at least ten microliters ofyeast suspension is placed onto the surface of the assimilation media.Multiple individual aliquots of yeast suspensions may be placed on eachminiature plate. The plates are then incubated at room temperature forabout 4 to 6 hours and thereafter checked for color change from purpleto yellow denoting carbohydrate assimilation.

An alternative embodiment of the miniaturized yeast assimilation systemhas been developed which combines prestarvation of the yeast inoculawith subsequent inoculation of the prestarved yeast onto a series ofminiaturized carbohydrate agars, incubation of said inocula on saidagars and post-incubation dye or fluorescent indicator addition fordetection of carbohydrate assimilation.

In this alternative embodiment, the yeasts were again taken from platescontaining a general yeast growth medium and suspended at aconcentration of McFarland No. 8, approximately 108 organisms, in eitherthe prestarvation media previously described or 1×10⁻⁵ M NaOH. Theprestarvation period may run from about 30 minutes to about 24 hourswith a preferred range from about 30 minutes to about 8 hours, and amost preferred prestarvation incubation period of about one hour at apreferred temperature of about 20° to 25° C. Prestarvations are, again,performed in sterile, sealed, non-glass containers at the previouslydescribed pH.

Following prestarvation, the carbohydrate depleted medium containing theyeast is vigorously mixed and, thereafter, at least 10 microliters ofyeast suspension is placed onto the surface of the assimilation medium.

The carbohydrate assimilation media employed in this alternativeembodiment were prepared by suspending appropriate amounts of yeastnitrogen base and agar into deionized water. The resulting mixture wasstirred with heat sufficient to dissolve the ingredients and the pH ofthe suspension was adjusted to about 7.2 with an allowable pH range offrom about 6.65 to about 7.35. The mixture was then sterilized at 121°C. at 15 psi for 15 minutes, cooled to about 40° to 55° C. and,thereafter, an appropriate amount of filter-sterilized carbohydrateadded aseptically. About 5 milliliter aliquots of this molten medium wasthen pipetted into sterile rectangular plates with attached flip-uplids, the plates having the approximate dimensions of 33×75×5millimeters. After solidification, the lids are closed and the plateswrapped in plastic lined aluminum foil pouches and stored at 4° C. Theseplates may be stored up to at least 8 months at 4° C. withoutdehydration. A nonexclusive list of the type of sugars which may beemployed in this alternative embodiment includes dextrose, galactose,sucrose, maltose, cellibiose, trehalose, lactose, melibiose, andraffinose. In the event that a single yeast inoculum is to besimultaneously tested for its ability to assimilate many differentcarbohydrates, the miniaturized plates shown in FIGS. 6 through 11 maybe employed. The individual wells such as 72a through 72h in FIG. 10 orcompartments such as 55a through 55h in FIG. 9 will each contain adifferent carbohydrate-containing medium and at least 10 microliters ofyeast suspension was distributed per individual well or compartment.

A preferred medium can be prepared following the procedure outlinedabove and employing from about 0.02 to about 0.7 weight percent yeastnitrogen base, from about 1.0 to about 5.0 weight percent agar, and fromabout 0.02 to about 1.0 weight percent carbohydrate, based on the amountof water added to these components. The preferred pH of the medium isfrom about 6.65 to about 7.35. A most preferred medium contains about0.067 weight percent yeast nitrogen base, about 2.0 weight percent agar,and about 0.1 weight percent carbohydrate, based on the amount of wateradded to these components. The most preferred pH is about 7.2.

Following inoculation of the carbohydrate-containing medium with anappropriate aliquot of yeast suspension, the plates are then coveredwith a sterile lid and incubated at room temperature for about 6 to 24hours. After incubatior, an appropriate amount of dye solution is addedto each assimilation test plate and assimilation detected by colorchanges in the dye. Three dyes were examined for possible use in thissystem, bromcresol purple, chlorophenol red and p-nitrophenol which givea purple to yellow, red to yellow and a colorless to yellow colorchange, respectively, when carbohydrate assimilation occurs.

The dyes were prepared by dissolving an appropriate amount of dye intowater, adjusting the pH of the dye solution to about 7.2 using 0.05 NNaOH, and filter-sterilizing the solution through a 0.22 μm filter. Abroad concentration range of dye solutions, from about 20 micrograms to250 micrograms bromcresol purple or chlorophenol red per milliliter orfrom about 15 micrograms to 45 micrograms p-nitrophenol per milliliter,may be employed as stock solutions from which an appropriate aliquot ofdye is taken and added to the carbohydrate assimilation test plates. Thepreferred concentration for the stock dye solutions is about 40micrograms bromcresol purple dye per milliliter, 60 mg chlorophenol reddye per milliliter and 20 micrograms p-nitrophenol dye per milliliter.The most preferred dye is bromcresol purple. Approximately 0.02 ml toabout 1.0 ml stock dye solution is added per about 0.1 to about 5.0milliliters of yeast inoculated carbohydrate assimilation test medium,respectively. The bromcresol purple reaction requires about 5 to 15minutes for completion, whereas the chlorophenol red requires about 15to 60 minutes for completion.

The miniaturized yeast identification system of the present inventionhas also been adapted to accommodate urea containing media. TheCryptococcus species, and occasional Trichosporon beigelii, and a veryrare Candida krusei possess the enzyme, urease, that is necessary forhydrolysis of urea. Presence of the urease enzyme in yeast cultures isindicated by color change of the medium from orange to pink.

The adaptation of urea containing medium to the miniaturized system ofthe present invention provides a positive detection of the enzyme,urease, in about one hour as opposed to the 24 to 72 hours and 3 to 4hours required for urease detection in presently available urea agarslants and urea broth, respectively. The shortened time-to-positivity ofthe present method is believed to be due to the increased organism tosubstrate ratio provided by the small media volume and increasedrelative surface area and by easier visualization of the color changeafforded by the shallower media depth in the miniaturized system ascompared to standard petri plates, agar slants, and broth tubes.

The miniature urease plates of the present invention are made bypreparing urea agar in accordance with manufacturer's instructions. Themedium was sterilized by heating to 121° C. at about 15 psi for 15minutes and is then dispensed in about 5 milliliter aliquots into theminiature plates described above. After solidification, the plates werewrapped and stored as described above so as to remain stable for atleast eight months when stored as described above. The plates wereinoculated by transferring one to three yeast colonies from a generalgrowth media onto the urease media. The plates were then allowed toincubate at 35° C. for one hour.

The media adapted for use in the miniaturized yeast identificationsystem of the present invention may be employed alone or in combination.When employing the partitioned plates shown in FIGS. 6 through 11, eachplate may, for example, contain a single type of medium inoculated withseveral different yeasts, or each plate may contain multiple, differentdifferential media inoculated with the same or multiple yeasts.

The fungal media of the present invention alone and in combinationprovide a rapid screening process for the identification of clinicallyimportant yeasts. Specifically, the miniaturized yeast identificationsystems described herein provide for the relatively rapid differentialidentification of Cryptococcus neoformans and the two Candida species,albicans and stellatoidea. It is further understood that while thisinvention has been described in relation to its preferred embodiments,the various modifications thereof will not be apparent to one skilled inthe art from reading this specification and it is intended to cover suchmodifications as fall within the scope of the appended claims.

EXAMPLES

The following examples demonstrate the mechanical advantages, increasedaccuracy and decreased time-to-positivity of the present invention overthose previously employed for the identification of various fungi,including Candida albicans, Candida stellatoidea and Cryptococcusneoformans. These examples are submitted for the purpose of providing abetter understanding of the present invention and are not to beconstrued as limiting the scope thereof.

EXAMPLE 1

This example was performed in order to compare the accuracy andsensitivity of egg white media, fetal bovine media, two commercialsystems, Flow GBE and A.P.I. GT™ Microtest, respectively, and theminiaturized SOC media of the present invention in detecting germ tubeformation. The ease of manipulation and microscopic examination of thesemedia was also compared.

Stock yeast strains were subcultured onto Sabouraud dextrose agar andallowed to grow for up to 72 hours. The yeast colonies formed by growthof the stock yeast on the Sabouraud dextrose agar were used to inoculatethe test media in this comparative study.

In both the egg white and serum media the substrate was added to a testtube, inoculated with yeast and incubated at 37° C. for 3 hours.Following incubation, a drop of yeast suspension was placed on amicroscope slide, covered with a glass coverslip, and examined under themicroscope at 100× to 400× magnification for germ tubes. Egg whitesubstrate was prepared by separating the egg white from the yolk of anegg. Fetal bovine serum substrate was purchased from Gibco. The Flow GBEtube, containing 0.1 percent weight per volume glucose and 2.6 percentweight per volume beef extract was inoculated with yeast and incubatedas per manufacturer's directions in a 35° to 37° C. incubator for 2 to 4hours. After incubation, one drop of inoculated broth was placed on amicroscope slide, covered with a glass coverslip, and examined under themicroscope at 100X magnification for germ tubes. The A.P.I. GT™Microtest, consisting of microtubes containing 70 microliterslyophilized rabbit plasma with EDTA, was prepared according tomanufacturer's directions, inoculated with yeast and incubated, as permanufacturer's instructions, in a 35° to 37° C. incubator for 2 to 5hours. After incubation, one drop of yeast suspension was placed on amicroscope slide, covered with a coverslip and examined under themicroscope at 400× magnification for the presence of germ tubes. Theminiaturized SOC plates were prepared as previously described. One tofour colonies of yeast are picked up with a sterile cotton swab and thebulk of the inoculum was deposited as a single mound onto theminiaturized SOC media. From the remaining inoculum on the swab, a thinfilm of yeast was streaked onto the medium in a dollar sign shape andthe thin film of yeast thereafter covered with a glass coverslip. Thelid of the miniaturized plate was closed, and the plate was incubated at37° to 40° C. for 3 hours. Following incubation, the miniaturized platewas opened, placed under the microscope, and the inoculum, centrallylocated under the coverslip, was examined at 100X to 400X magnificationfor germ tubes.

Table 1 gives the comparative results on the performance of germ tubesystems. Twenty strains of Candida albicans were tested for germ tubeformation and ease of microscopic examination on egg white, fetal bovineserum, Flow GBE, A.P.I. GT™ Microtest and the miniaturized SOC medium ofthe present invention. Each strain was examined after three hours ofincubation and marked as positive (+) for germ tube formation when atleast eight out of ten microscopic fields showed germ tube formation.The strains were marked as marginal (±) when germ tubes were visible inonly one or two out of ten microscopic fields. Clumping was said tooccur when, despite agitation, the germinated cells remained knottedtogether in masses, making it difficult to observe, microscopically,whether there is true germ tube or pseudohyphal formation.

                  TABLE 1                                                         ______________________________________                                        COMPARISON OF THE MINIATURIZED SOC PLATE                                      WITH OTHER AVAILABLE SYSTEMS FOR GERM TUBE                                    FORMATION BY Candida albicans                                                              Germ Tube Formation                                                      No. of +         ±      Clumping                                   System    Isolates No.    %    No.  %    No.  %                               ______________________________________                                        1.  SOC       20       20   100  0    0    0     0                            2.  Egg white 20       20   100  0    0    0     0                            3.  Fetal bo- 20       20   100  0    0    2    10                                vine serum                                                                4.  Flow GBE  20       18    90  2    10   4    20                                tube                                                                      5.  API GT    20       17    85  3    15   16   80                                Microtest                                                                 ______________________________________                                    

As can be seen from a study of Table 1, 100 percent of the strainstested formed germ tubes in three hours on the miniaturized SOC, eggwhite and fetal bovine serum media, while only 90 percent and 85 percentof the strains formed germ tubes in the Flow GBE and A.P.I. GT™Microtest system, respectively. As shown in the results displayed inTable 1, significant clumping was observed in the serum, Flow GBE andA.P.I. GT™ Microtest systems. The miniaturized SOC medium is a solidsupport medium on which yeast cells are dispersed in a thin filmallowing observation of single stationary cells. In the other systems,the yeasts are usually in motion in the liquid under the coverslip,making it difficult to focus on any one cell.

From the standpoint of the manipulation, the miniaturized SOC platerequires only an initial inoculum transfer, whereas the other systemsrequire a second transfer to a microscope slide which increasestechnician time and necessitates additional materials.

EXAMPLE 2

This example was performed in order to establish the effect of variousconcentrations of the phenol oxidase substrate, caffeic acid, on yeastmorphology and pigment formation on SOC media in the miniaturizedsystem. The specific morphologies observed were germ tube,chlamydospore, blastospore, filaments and pseudohyphae formation. Thebrown pigment production characteristic of several Cryptococcusneoformans strains was also monitored.

The test medium, the miniaturized SOC medium, of the present example wasprepared as follows: 20 grams of agar, 10 grams of oxgall, 10 grams ofpurified saponin, 0.54 grams of ammonium chloride, and various amountsof caffeic acid, listed in Table 2 below, were added to one liter ofdeionized water. The mixture thus formed was stirred and heated toboiling to dissolve the ingredients and adjusted to a final pH of about7.2. The solution is then sterilized by heating to about 121° C. at 15psi for 15 minutes. After cooling, the medium was dispensed to a depthof about 2 millimeters (approximately 5 milliliters) into sterile,miniature plates, previously described, and allowed to solidify.

One to four yeast colonies were transferred from a general growth mediumto the test medium, as described in Example 1. The inoculated plateswere incubated for 3 hours at 37° C. and thereafter at room temperature.

Table 2 sets forth the results of the test performed in order to comparethe effect of various concentrations of caffeic acid on yeast morphologyand pigment production on the miniaturized SOC medium. Six species ofCandida and four species of Cryptococcus were plated on the test mediumand observed at the time interval specified in Table 2. Microscopicexamination at the time intervals designated in Table 2 was conducted at100× magnification in the manner described in Example 1. Positivemorphology (+) was defined as at least 8 out of 10 microscopic fieldshaving shown a particular morphology or combination of morphologies.Negative morphology (-) was defined as less than 1 or 2 out of 10microscopic fields having shown a particular anticipated morphology orcombination thereof.

As can be seen from Table 2 below, the miniaturized SOC media supportedthe characteristic yeast morphologies and pigment production at arelatively wide range of caffeic acid concentrations, from about 0.005weight percent to about 0.5 weight percent. In pigment formation, 10percent of the Cryptococcus neoformans strains tested produced browncoloration at 18 hours on SOC with 0.006 weight percent caffeic acid. At0.6 weight percent, SOC itself was dark brown, making the yeast's colorchange difficult to recognize and, also, the SOC medium became a darkerbrown color each day, indicating instability. The effect of caffeic acidconcentration on general yeast morphology, as shown in Table 2 below,establishes an upper limit for caffeic acid. Essentially allfilamentation of Candida species was inhibited at 0.6 weight percentcaffeic acid.

                                      TABLE 2                                     __________________________________________________________________________    MORPHOLOGY AND PIGMENT PRODUCTION ON MINIATURIZED                             SOC MEDIUM AT VARIOUS CONCENTRATIONS OF CAFFEIC ACID                                                   Weight Percent Caffeic Acid                          Organism     Incubation Time (hrs.)                                                                    0.006 0.06  0.12  0.6                                __________________________________________________________________________    Candida albicans                                                                            3          gt+   gt+   gt-+  gt-                                             24          fil +/cm+                                                                           fil +/cm+                                                                           fil +/cm+                                                                           fil -/cm-                          Candida stellatoidea                                                                        3          gt-   gt-   gt-   gt-                                             24          fil +/cm-                                                                           fil +/cm-                                                                           fil +/cm-                                                                           fil -/cm-                          Candida tropicalis                                                                          3          ps+   ps+   ps-   ps-                                             24          fil+  fil+  fil+  fil-                               Candida krusei                                                                              3          b+    b+    b+    b+                                              24          fil+  fil+  fil+  fil-                               Candida guilliermondii                                                                      3          b+    b+    b+    b+                                              24          fil+  fil+  fil+  fil-                               Candida parapsilosis                                                                        3          b+    b+    b+    b+                                              24          fil+  fil+  fil+  fil-                               Cryptococcus neoformans*                                                                   18          brn   brn   brn   brn                                Cryptococcus laurentii                                                                     18          crm   crm   crm   crm                                Cryptococcus albidus                                                                       18          crm   crm   crm   crm                                Cryptococcus diffluens                                                                     18          crm   crm   crm   crm                                __________________________________________________________________________     *Twenty (20) strains were tested                                              gt = germ tube; fil = filament; cm = chlamydospore, ps = pseudohyphae b =     blastospore, brn = brown, crm = cream                                    

EXAMPLE 3

This example was performed to determine the effects of ammonium chlorideaddition to SOC medium on chlamydospore production by Candida albicans.

The test medium of the present example was prepared as follows: 20 gramsof agar, 10 grams of oxgall, 10 grams of purified saponin, 0.6 gramscaffeic acid, and varying concentrations of ammonium chloride, indicatedin Table 3 below, were added to one liter of deionized water. Themixture was then stirred, heated to boiling, adjusted to a final pH ofabout 7.2, and sterilized by heating to 121° C. at 15 psi for 15minutes. After a cooling period, about 5 milliliters of cooled mediumwas dispensed into the miniature plates as described in Example 1. Theseplates may be stored up to 8 months when stored as described in Example1.

One to four yeast colonies grown on Sabouraud dextrose agar were platedas described in Example 1 on SOC medium containing the variousconcentrations of ammonium chloride listed in Table 3 below. At 1.0 Mammonium chloride, some of the medium components precipitated making itimpossible to test that formulation. The yeast colonies so platedcomprised 20 different strains of Candida albicans which had, onoccasion, given negative or weak chlamydospore production. The plateswere incubated for 3 hours at 37° C. and examined for germ tubeformation. All 20 strains produced numerous germ tubes on allconcentrations of ammonium chloride listed in Table 3. The plates werethen incubated at room temperature for 24 to 72 hours and examined forchlamydospore production by the methods described in Example 1 and atthe time intervals indicated in Table 3 below.

As can be seen by examination of Table 3, the addition of ammoniumchloride significantly enhanced chlamydospore production. The optimumtime for determining chlamydospore production was 24 hours to 48.

                  TABLE 3                                                         ______________________________________                                        EFFECTS ON NH.sub.4 Cl ADDITION TO SOC ON                                     CHLAMYDOSPORE PRODUCTION IN TWENTY STRAINS                                    OF Candida albicans                                                           Chlamydospore Production                                                      NH.sub.4 Cl, Molarity                                                                         0        0.001   0.01   0.1                                   Incubation                                                                            No. of  %        %       %      %                                     Time (hrs.)                                                                           Strains Positive Positive                                                                              Positive                                                                             Positive                              ______________________________________                                        24      20      45.0      80.0   100.0  50.0                                  48      20      60.0     100.0   100.0  80.0                                  72      20      100.0    100.0   100.0  100.0                                 ______________________________________                                    

EXAMPLE 4

This example was performed in order to test the ability of theminiaturized carbohydrate assimilation media of the present invention,performed by the method of the present invention, to differentiatebetween Candida albicans and Candida stellatoidea.

The miniaturized sucrose assimilation media employed in this example wasprepared by adding 20 grams of agar, 20 milligrams of bromcresol purple,0.67 grams yeast nitrogen base and 4 milliliters of 0.5 N sodiumhydroxide to one liter of deionized water and adjusting to a pH of about7.2. The media was sterilized by heating to about 121° C. at 15 psi for15 minutes and after cooling, presterilized sucrose is addedasceptically to yield a final sucrose concentration of 1.0 percent. Themedia was then dispensed in about 5 milliliter aliquots into theminiature plates, as described above. These plates may be stored, by themethod described in Example 1, up to eight months without dehydration orloss of color.

Six species of Candida were first grown on Sabouraud dextrose agarcontaining gentamicin, as previously described. Yeasts were taken fromthis primary growth medium and suspended in sterile deionized water andheld for at least one hour at room temperature for purposes ofprestarvation. After prestarvation, the suspension was vortexed and,using a sterile pipet, one to about three drops of yeast suspension wasplaced onto the surface of the miniaturized sucrose media. The plateswere then incubated at room temperature for 4 to 6 hours. Following theincubation period the plates were checked for color change from purpleto yellow indicating sucrose assimilation. The plates were held at roomtemperature for 24 hours and read a second time.

The results of this example are shown in Table 4A below. Candidaalbicans, Candida stellatoidea, Candida tropicalis, Candida krusei andCandida glabrata gave the correct assimilation response on theminiaturized sucrose assimilation plates after 5 hours incubation atroom temperature and maintained the correct response for 24 hours.Candida parapsilosis, however, did not show a characteristic positivereaction until 18 to 24 hours. From this data, it is apparent thatCandida albicans and Candida stellatoidea can be differentiated in 5hours.

                                      TABLE 4A                                    __________________________________________________________________________    EXAMINATION OF THE MINIATURE SUCROSE                                          ASSIMILATION PLATE WITH VARIOUS YEASTS                                                         Sucrose Assimilation                                                          5 hr.        24 hr.                                                 No. of                                                                             Correct                                                                            +      -     +      -                                        Organism                                                                             Isolates                                                                           Response                                                                           No.                                                                              %   No.                                                                              %  No. %  No.                                                                              %                                     __________________________________________________________________________    C. albicans                                                                          120  +    120                                                                              100.0                                                                              0  0.0                                                                             120 100.0                                                                             0  0.0                                  C. stellatoidea                                                                      21   -    0  0.0 21 100.0                                                                            0    0.0                                                                             21 100.0                                 C. tropicalis                                                                        29   +    29 100.0                                                                              0  0.0                                                                             29  100.0                                                                             0  0.0                                  C. parapsilosis                                                                      31   +    0  0.0 31 100.0                                                                            31  100.0                                                                             0  0.0                                  C. krusei                                                                            18   -    0  0.0 18 100.0                                                                            0    0.0                                                                             18 100.0                                 C. glabrata                                                                          34   -    0  0.0 34 100.0                                                                            0    0.0                                                                             34 100.0                                 __________________________________________________________________________

To further pursue the concept of miniature assimilation for othercarbohydrates besides sucrose, identical experiments were performedwhere media were prepared as described above except that 1 gram of eachsugar listed below in Table 4B was substituted for sucrose to yield aspecific sugar containing medium. Yeasts were inoculated, plated andgrown as described above and examined for carbohydrate assimilation atthe time indicated in Table 4B below.

As shown in Table 4B, the proper assimilation pattern for each speciescan be achieved within 24 hours. With Candida parapsilosis, there was adiscrepancy with the positive sucrose assimilation at 5 hours and thenegative results in the previous experiment as given in Table 4A. Thisis a significant example of strain variation among yeast strains.

                                      TABLE 4B                                    __________________________________________________________________________    EXAMINATION OF MINIATURE CARBOHYDRATE ASSIMILATION                            PLATES WITH VARIOUS YEASTS                                                    Assimilation                                                                                           5 hr.         24 hr.                                        No. of       Correct                                                                            +      -      +     -                                Organism                                                                             Isolates                                                                           Carbohydrate*                                                                         Response                                                                           No.                                                                              %   No.                                                                              %   No.                                                                              %  No.                                                                              %                             __________________________________________________________________________    C. albicans                                                                          3    suc     +    3  100.0                                                                             0  0.0 3  100.0                                                                            0  0.0                                       malt    +    3  100.0                                                                             0  0.0 3  100.0                                                                            0  0.0                                       galac   +    3  100.0                                                                             0  0.0 3  100.0                                                                            0  0.0                                       treh    +    2  66.7                                                                              1  33.3                                                                              3  100.0                                                                            0  0.0                                       lac     -    0  0.0 3  100.0                                                                             0   0.0                                                                             3  100.0                         C. stellatoidea                                                                      3    suc     -    0  0.0 3  100.0                                                                             0   0.0                                                                             3  100.0                                     malt    +    3  100.0                                                                             0  0.0 3  100.0                                                                            0  0.0                                       galac   +    3  100.0                                                                             0  0.0 3  100.0                                                                            0  0.0                                       treh    +    0  0.0 3  100.0                                                                             3  100.0                                                                            0  0.0                                       lac     -    0  0.0 3  100.0                                                                             0   0.0                                                                             3  100.0                         C. tropicalis                                                                        3    suc     +    3  100.0                                                                             0  0.0 3  100.0                                                                            0  0.0                                       malt    +    3  100.0                                                                             0  0.0 3  100.0                                                                            0  0.0                                       galac   +    1  33.3                                                                              2  66.7                                                                              3  100.0                                                                            0  0.0                                       treh    +    1  33.3                                                                              2  66.7                                                                              3  100.0                                                                            0  0.0                                       lac     -    0  0.0 3  100.0                                                                             0   0.0                                                                             3  100.0                         C. parapsilosis                                                                      3    suc     +    3  100.0                                                                             0  0.0 3  100.0                                                                            0  0.0                                       malt    +    1  33.3                                                                              2  66.7                                                                              3  100.0                                                                            0  0.0                                       galac   +    2  66.7                                                                              1  33.3                                                                              3  100.0                                                                            0  0.0                                       treh    +    0  0.0 3  100.0                                                                             3  100.0                                                                            0  0.0                                       lac     -    0  0.0 3  100.0                                                                             0   0.0                                                                             3  100.0                         __________________________________________________________________________     *suc = sucrose; malt = maltose; galac = galactose; treh = trehalose; lac      lactose                                                                  

EXAMPLE 5

This example was performed to test the ability of miniaturized ureasemedia to detect the presence of the urease enzyme in various species ofCryptococcus.

The miniature urease plates in the present example were made bypreparing urea agar according to manufacturer's instructions,sterilizing by heating at 121° C. at 15 psi for 15 minutes and thendispensing the media into the plates as described in the previousexamples. Again, by storing as described in Example 1, the plates remainstable for up to at least 8 months.

The plates were inoculated with four species of Cryptococcus and sixspecies of Candida, by transferring, with a sterile cotton swab, two tothree yeast colonies from a general growth medium such as Sabourauddextrose agar. Two to three yeasts of a single species were inoculatedper plate and the plates were incubated at 35° C. for up to 24 hours.

The results of this example are shown in Table 5. The Cryptococcusspecies were positive in one hour and the other yeast isolates remainednegative for 24 hours. A hydrolysis of urea releases ammonium gas whichis basic and rapidly spreads throughout the medium turning the mediumfrom orange to pink. A positive organism completely changes the mediumcolor in the miniature plate within two to three hours. Therefore, ifmore than one organism is inoculated onto the miniaturized urease plate,the plate must be read between about one and two hours or false positivereactions can occur.

                  TABLE 5                                                         ______________________________________                                        EXAMINATION OF THE MINIATURE UREASE PLATE                                     WITH VARIOUS YEASTS                                                                             Urease Hydrolysis*                                                  No. of Correct  +          -                                          Organism  Isolates Response No.  %     No.  %                                 ______________________________________                                        Cr. neoformans                                                                          81       +        81   100.00                                                                               0    0.0                              Cr. albidus                                                                             20       +        20   100.0  0    0.0                              Cr. laurentii                                                                           10       +        10   100.00                                                                               0    0.0                              Cr. diffluens                                                                           24       +        24   100.00                                                                               0    0.0                              C. albicans                                                                             122      -        0    0.0   122  100.0                             C. stellatoidea                                                                         21       -        0    0.0   21   100.0                             C. tropicalis                                                                           29       -        0    0.0   29   100.0                             C. parapsilosis                                                                         32       -        0    0.0   32   100.0                             C. krusei 18       -(+)**   0    0.0   18   100.0                             C. glabrata                                                                             34       -        0    0.0   34   100.0                             ______________________________________                                         *The reactions were read at one hour and negative reactions were              reconfirmed at 24 hours.                                                      **C. krusei has been reported to rarely show a positive urease reaction,      but the strains used in this experiment were negative.                   

EXAMPLE 6

This example was performed to compare the post-incubation addition ofdye for detection of carbohydrate assimilation with conventionalcarbohydrate assimilation methods. The carbohydrate assimilationsdetected by post-incubation addition of dye were conducted inMicrotiter™ plates and are, hence, referred to as "Microtiter™carbohydrate agar assimilation" in the text and Tables 6A and 6B, below.

The Microtiter™ carbohydrate assimilation media employed in this examplewere prepared by adding 20 grams of agar and 0.67 grams of yeastnitrogen base to 950 milliliters deionized water. The mixture wasstirred with heat to dissolve the ingredients, adjusted to a pH of about7.2 with 0.5 N NaOH, divided into eight equal parts, sterilized at 121°C. at 15 psi for 15 minutes and, thereafter, allowed to cool to about45° to 55° C. Then, 6.25 milliliters of a two percent, filtersterilized, carbohydrate solution containing one of each of the eightcarbohydrates listed in Table 6A or 6B, below, was asceptically added toone of each of the cooled solutions. The two percent carbohydratesolutions were prepared by adding 0.2 grams of carbohydrate to 10milliliters deionized water and adjusting to a pH of about 7.0 with 0.05N NaOH. After mixing, 0.15 milliliters aliquots of thecarbohydrate-containing medium was sterily dispensed into the wells of aMicrotiter™ plate.

The yeast listed in Tables 6A and 6B, below, were taken from platescontaining a general yeast growth medium as described in Example 4 andsuspended at a concentration of McFarland No. 8, approximately 10⁸organisms, in 1×10⁻⁵ M NaOH and held for about one hour at roomtemperature for purposes of prestarvation. Following prestarvation, thesuspension was vortexed and 0.02 milliliter aliquots were asepticallytransferred into each medium containing well of the Microtiter™ plate.The plates were covered with a sterile lid and incubated at roomtemperature for 24 hours. After incubation, 0.02 milliliter aliquots offilter sterilized, 20 micrograms bromcresol purple per milliliter dyesolution was added to each well of the Microtiter™ plates. The plateswere read 5 to 10 minutes after dye addition for a purple to yellowcolor change indicative of positive assimilation of carbohydrate.

The results of the Microtiter™ carbohydrate agar assimilations are givenin Tables 6A and 6B, below, wherein the data are compared to reports forconventional methods given in The Yeasts by Lodder and the A.P.I. 20CStrip. The conventional methods include, Wickerham liquid, Saito agar,and auxanographic technique.

                                      TABLE 6A                                    __________________________________________________________________________    COMPARISON OF MICROTITER ™ CARBOHYDRATE AGAR ASSIMILATION                  WITH REPORTS FROM CONVENTIONAL ASSIMILATION METHODS*                                       No. of      Assimilation at 24 hrs.**                            Organism     Isolates                                                                           System dextrose                                                                           galactose                                                                          sucrose                                                                           maltose                                                                            cellibiose                                                                         trehalose                                                                          lactose                                                                           raffinose           __________________________________________________________________________    C. albicans  --   Conventional                                                                         +    +    +   +    -    +    -   -                                45   Microtiter                                                                           +    +    +   +    -    +    -   -                   C. stellatoidea                                                                            --   Conventional                                                                         +    +    -   +    -    +    -   -                                12   Microtiter                                                                           +    +    -   +    -    +    -   -                   C. tropicalis                                                                              --   Conventional                                                                         +    +    +   +    v    +    -   -                                32   Microtiter                                                                           +    +    +   +    ∓ +    -   -                   C. parapsilosis                                                                            --   Conventional                                                                         +    +    +   +    -    +    -   -                                12   Microtiter                                                                           +    +    +   +    -    +    -   -                   C. krusei    --   Conventional                                                                         +    -    -   -    -    -    -   -                                 8   Microtiter                                                                           +    -    -   -    -    -    -   -                   C. glabrata  --   Conventional                                                                         +    -    -   -    -    +    -   -                                16   Microtiter                                                                           +    -    -   -    -    +    -   -                   Cr. neoformans                                                                             --   Conventional                                                                         +    +    +   +    w    +    -   w                                 8   Microtiter                                                                           +    +    +   +    ∓ ± -   -                   Cr. laurentii                                                                              --   Conventional                                                                         +    +    +   +    +    +    +   +                                 2   Microtiter                                                                           +    +    +   +    +    +    +   +                    Cr. albidus var. diffluens                                                                --   Conventional                                                                         +    w    +   +    +    +    w   w                                 7   Microtiter                                                                           +    ∓ +   +    ± +    -   -                   __________________________________________________________________________     *Conventional Methods: Wickerham liquid method, Saito agar method,            auxanographic method.                                                         **v = variable; w = weak; ± = ≧50%; ∓ = ≦50%         

                                      TABLE 6B                                    __________________________________________________________________________    COMPARISON OF MICROTITER ™ CARBOHYDRATE AGAR                               ASSIMILATION WITH A.P.I. 20C STRIP REPORTS                                            Total                                                                         No.     Assimilation at 24 hrs.                                               of Iso- dextrose                                                                           galactose                                                                           sucrose                                                                             maltose                                                                             cellibiose                                                                          trehalose                                                                           lactose                                                                             raffinose            Organism                                                                              lates                                                                             System                                                                            No.  %                                                                             No.                                                                              %  No.                                                                              %  No.                                                                              %  No.                                                                              %  No.                                                                              %  No.                                                                              %  No.                                                                              %                 __________________________________________________________________________    C. albicans                                                                            --*                                                                              API -- 100                                                                             -- 100                                                                              --  96                                                                              -- 100                                                                              -- 0  --  99                                                                              -- 0  -- 0                         45  Micro-                                                                            45 100                                                                             45 100                                                                              45 100                                                                              45 100                                                                              0  0  45 100                                                                              0  0  0  0                             titer                                                             C. stellatoidea                                                                       --  API -- 100                                                                             -- 100                                                                              --  0 -- 100                                                                              -- 0  --  1 -- 0  -- 0                         12  Micro-                                                                            12 100                                                                             12 100                                                                              0   0 12 100                                                                              0  0  12 100                                                                              0  0  0  0                             titer                                                             C. tropicalis                                                                         --  API -- 100                                                                             --  99                                                                              -- 100                                                                              -- 100                                                                              -- 12 -- 100                                                                              -- 0  -- 0                         32  Micro-                                                                            32 100                                                                             32 100                                                                              32 100                                                                              31  97                                                                              15 47 32 100                                                                              0  0  0  0                             titer                                                             C. parapsilosis                                                                       --  API -- 100                                                                             -- 100                                                                              -- 100                                                                              -- 100                                                                              -- 0  --  97                                                                              -- 0  -- 0                         12  Micro-                                                                            12 100                                                                             12 100                                                                              12 100                                                                              12 100                                                                              0  0  12 100                                                                              0  0  0  0                             titer                                                             C. krusei                                                                             --  API -- 100                                                                             --  0 --  0 --  0 -- 0  --  0 -- 0  -- 0                          8  Micro-                                                                             8 100                                                                              0  0 0   0  0  0 0  0   0  0 0  0  0  0                             titer                                                             C. glabrata                                                                           --  API -- 100                                                                             --  0 --  0 --  0 -- 0  --  99                                                                              -- 0  -- 0                         16  Micro-                                                                            16 100                                                                              0  0 0   0  0  0 0  0  16 100                                                                              0  0  0  0                             titer                                                             Cr. neoformans                                                                        --  API -- 100                                                                             --  99                                                                              -- 100                                                                              -- 100                                                                              -- 37 --  73                                                                              -- 0  -- 86                         8  Micro-                                                                             8 100                                                                              8 100                                                                              8  100                                                                               8 100                                                                              1  13  4  50                                                                              0  0  0  0                             titer                                                             Cr. laurentii                                                                         --  API -- 100                                                                             -- 100                                                                              -- 100                                                                              --  92                                                                              -- 92 --  92                                                                              -- 99 -- 99                         2  Micro-                                                                             2 100                                                                              2 100                                                                              2  100                                                                               2 100                                                                              2  100                                                                               2 100                                                                              2  100                                                                              2  100                           titer                                                             Cr. albidus                                                                           --  API -- 100                                                                             --  0 -- 100                                                                              --  99                                                                              -- 99 --  93                                                                              -- 0  -- 68                var. diffluens                                                                         7  Micro-                                                                             7 100                                                                              2  29                                                                              7  100                                                                               7 100                                                                              6  86  7 100                                                                              0  0  0  0                             titer                                                             __________________________________________________________________________     *Total number of isolates given only in A.P.I. databank.                 

As shown in Table 6A above, there is good correlation between theMicrotiter™ carbohydrate agar assimilation test and the publishedresults of conventional Wickerham liquid, Saito agar and auxanographicmethods. Certain carbohydrate reactions with Cr. neoformans and Cr.albidus, however, require examination.

As shown in Table 6B, above, there is an overall good correlationbetween the Microtiter™ carbohydrate agar assimilation test and theA.P.I. 20C strip method with the exceptions of raffinose assimilationfor Cr. neoformans and Cr. albidus.

EXAMPLE 7

This example was performed in order to test the effect of an increasedratio of surface area of organisms to volume of medium on thetime-to-positivity in carbohydrate assimilation. One way to demonstratethe effect of said increased ratio is to hold the surface area andmicrobial inoculum constant while varying the volume, herein expressedas depth of medium, in a given test plate or tube.

Three different containers were used to examine the effect of surfacearea to volume ratios on time-to-positivity: the miniaturized cultureplates of the present invention, standard 100×15 millimeters petriplates, and standard 16×125 millimeter flat-bottom culture tubes. Thetheoretical limits of the surface area to volume ratio employed in agiven container will be set by the dimensions necessary to visualize thecolor change prompted by positive carbohydrate assimilation and aminimal depth necessary to preclude dehydration of the medium.

Sucrose assimilation medium prepared as described in Example 4, above,was aseptically poured into each of the containers at the depths shownin FIG. 13.

Candida albicans, the test organism, was first grown on Sabouranddextrose agar containing gentamicin, as previously described. Yeastswere taken from this primary growth medium and suspended in steriledeionized water and held at least one hour at room temperature forpurposes of prestarvation. Following prestarvation, the suspension wasvortexed and, using a sterile pipet, one to about three drops of yeastsuspension was placed onto the surface of each miniaturized plate andculture tube containing sucrose medium and three one-drop inoculationsof yeast were placed on each 100×15 millimeter (mm) petri platecontaining sucrose medium. The plates and tubes were then incubated atroom temperature for not more than 12 hours. Each plate or tube of thedepth specified in the graph in FIG. 13, was run in duplicate. Positivereactions, denoting sucrose assimilation, were determined by an obviouscolor change from purple to yellow at the post-inoculation timeintervals shown in the graph in FIG. 13, given as "Time-to-Positivity"in hours.

The results of this example are shown in the graph in FIG. 13. Thisexample confirms that at a medium depth range of 2 to 2.5 mm,approximately 4 to 5 milliliters of medium per plate, in theminiaturized plates (0--0), the time-to-positivity for sucroseassimilation by Candida albicans is from about four to five hours.Although a shift in the time-to-positivity is seen with increasing depthor volume at a constant surface area of organisms in the miniaturizedplates, the most dramatic effect of increased medium depth or volume ata constant surface area of organisms is seen in the standard 100×15 mmpetri plates (Δ--Δ). Additionally, the medium depth in the standardpetri plate is typically 5 mm corresponding to 18 to 20 milliliters ofmedium per 100×15 mm plate which is shown to yield about a 10 hourtime-to-positivity for Candida albicans in the sucrose assimilationtest. Furthermore, medium depths of 3 mm or less in the 100×15 mm petriplates are not preferred due to possible problems with dehydrationduring storage. The greater time-to-positivity seen in the 16×125 mmflat-bottom culture tubes ( -- ) is thought to be due to problems withoxygen tension created by the closed environment of the culture tubes ascompared to the open system of the plates.

We claim:
 1. A culture container of a size to fit on a conventionalmicroscope viewing stand for the identification of fungal pathogens,comprising:(a) a well means for containing fungal differentiation media,the dimensions of said well means adapted to contain a quantity of up toabout 5.0 milliliters of media at a depth of up to about 4.5millimeters, for the growth of a specimen; and (b) a fungaldifferentiation media present in said well means in the amount of saidquantity comprising a composition effective to induce a differentobservable effect in at least one species of fungi as compared withanother species of fungi and effective to induce germ tube productionand conventional morphological characteristics, said compositionincluding purified saponin, oxgall, a substrate for phenol oxidase insufficient quantity to allow differentiation of Cryptococus neoformans,water and ammonium ions.
 2. The culture container of claim 1, furthercomprising a lid adapted to removable engage with said container.
 3. Theculture container of claim 1, wherein said fungal differentiation mediafurther comprises a carrying agent.
 4. The culture container of claim 1,wherein said substrate for phenol oxidase is caffeic acid.
 5. Theculture container of claim 1, wherein said substrate for phenol oxidasecomprises from about 0.005 to about 0.5 weight percent of the weight ofsaid water.
 6. The culture container of claim 5, wherein said substratefor phenol oxidase comprises from about 0.012 to about 0.12 weightpercent of the weight of said water.
 7. The culture container of claim6, wherein said substrate for phenol oxidase comprises about 0.06 weightpercent of the weight of said water.
 8. The culture container of claim1, wherein said saponin comprises from about 0.1 to about 5.0 weightpercent of the weight of said water.
 9. The culture container of claim8, wherein said saponin comprises from about 0.1 to about 2.5 weightpercent of the weight of said water.
 10. The culture container of claim9, wherein said saponin comprises about 1.0 weight percent of the weightof said water.
 11. The culture container of claim 3, wherein saidcarrying agent is present in an amount of from about 1.0 to about 5.0weight percent of the weight of said water.
 12. The culture container ofclaim 11, wherein said carrying agent is present in an amount of 2.0weight percent of the weight of said water.
 13. The culture container ofclaim 3, wherein said carrying agent is agar.
 14. The culture containerof claim 1, wherein said oxgall is present in an amount from about 0.25to about 30.0 weight percent of the weight of said water.
 15. Theculture container of claim 14, wherein said oxgall is present in anamount from about 0.5 to about 5.0 weight percent of the weight of saidwater.
 16. The culture container of claim 15, wherein said oxgall ispresent in an amount of about 1.0 weight percent of the weigth of saidwater.
 17. The culture container of claim 1, wherein said ammonium ionsare present in an amount from 0.001 M to about 0.5 M.
 18. The culturecontainer of claim 17, wherein said ammonium ions are present from about0.005 M to about 0.05 M.
 19. The culture container of claim 18, whereinsaid ammonium ions are present at about 0.01 M.
 20. The culturecontainer of claim 1, wherein said well means is cylindrical.
 21. Theculture container of claim 1, wherein said well means is pie-shaped. 22.The culture container of claim 1, wherein said well means has aperimeter of rectangular or square shape.
 23. The culture container ofclaim 1, further comprising at least one additional well means forcontaining fungal differentiation media.
 24. The culture container ofclaim 1, wherein said quantity is from about 2.5 milliliters to about5.0 milliliters at a depth of up to about 4.5 millimeters.
 25. Theculture container of claim 23 further comprising at least one additionalmedia for fungal differentiation or growth, each of said additionalmedias situated in one of said additional well means.
 26. The culturecontainer of claim 23, wherein a second fungal differentiation media ispresent in at least one of said additional well means in the amount ofsaid quantity comprising a media effective to induce a differentobservable effect in at least one species of fungi subjected to aprevious carbohydrate depletion step as compared to another species offungi subjected to said carbohydrate depletion step, wherein said secondfungal differentiation media is adapted in composition to produce anobservable effect of positive or negative carbohydrate assimilation incarbohydrate depleted fungal cells within from about 3 hours to about 24hours from inoculation.
 27. The culture container of claim 23 wherein asecond fungal differentiation media is present in at least one of saidadditional well means in the amount of said quantity comprising a mediaadapted in composition to induce a different observable effect in afungal species metabolizing urea as compared to a fungal species whichdoes not within from about 1 hour to about 3 hours after inoculation.28. The culture container of claim 23 wherein a second fungaldifferentiation media is present in at least one of said additional wellmeans in the amount of said quantity comprising a media effective toinduce a different observable effect in at least one species of fungisubjected to a previous carbohydrate depletion step as compared toanother species of fungi subjected to said carbohydrate depletion step,wherein said second fungal differentiation media is adapted incomposition to produce an observable effect to positive or negativecarbohydrate assimilation in carbohydrate depleted fungal cells withinfrom about 3 to about 24 hours from inoculation, and a third fungaldifferentiation media is present in another of said additional wellmeans adapted in composition to induce a different observable effect ina fungal species metabolizing urea as compared to a fungal species whichdoes not within from about 1 hour to about 3 hours after inoculation.29. A culture container of a size to fit on a conventional microscopeviewing stand for the identification of fungal pathogens, comprising:(a)a well means for containing fungal differentiation media, the dimensionsof said well means adapted to contain a quantity of up to about 5.0milliliters of media at a depth of up to about 4.5 millimeters, for thegrowth of a specimen; and (b) a fungal differentiation media present insaid well means in the amount of said quantity, comprising about 1.0weight percent purified saponin, about 1.0 weight percent oxgall, about0.06 weight percent caffeic acid, about 0.01 M ammonium ions, about 2.0weight percent agar, and water, said weight percentages based on theweight of said water.
 30. A culture container of a size to fit on aconventional microscope viewing stand for the identification of fungalpathogens, comprising:(a) at least two well means for containing fungaldifferentiation media, the dimensions of each of said two well meansadapted to contain a quantity of up to about 5.0 milliliters of media ata depth of up to about 4.5 millimeters, each of said well means for thegrowth of a specimen; and (b) a first fungal differentiation mediapresent in one of said well means in the amount of said quantitycomprising purified saponin, oxgall, a substrate for phenol oxidase,ammonium ions, a carrying agent, and water, and a second fungaldifferentiation media present in the other of said well means in theamount of said quantity comprising a media effective to induce adifferent observable effect in at least one species of fungi subjectedto a previous carbohydrate depletion step as compared to another speciesof fungi subjected to said carbohydrate depletion step, wherein saidsecond fungal differentiation media is adapted in composition to producean observable effect of positive or negative carbohydrate assimilationin carbohydrate depleted fungal cells within from about 3 hours to about24 hours from inoculation.
 31. The culture container of claim 30,further comprising a lid adapted to removably engage with saidcontainer.
 32. The culture container of claim 30, wherein said quantityis from about 2.5 milliliters to about 5.0 milliliters at a depth of upto about 4.5 millimeters.
 33. A culture container of a size to fit on aconventional microscope viewing stand for the identification of fungalpathogens, comprising:(a) at least two well means for containing fungaldifferentiation media, the dimensions of each of said two well meansadapted to contain a quantity of up to about 5.0 milliliters of media ata depth of up to about 4.5 millimeters, each of said well means for thegrowth of a specimen; and (b) a first fungal differentiation mediapresent in one of said well means in the amount of said quantitycomprising purified saponin, oxgall, a substrate for phenol oxidase,ammonium ions, a carrying agent, and water, and a second fungaldifferentiation media present in the other of said well means in theamount of said quantity comprising a media adapted in composition toinduce a different observable effect in a fungal species metabolizingurea as compared to a fungal species which does not within from about 1hour to about 3 hours after inoculation.
 34. The culture container ofclaim 33, further comprising a lid adapted to removably engage with saidcontainer.
 35. The culture container of claim 33, wherein said quantityis from about 2.5 milliliters to about 5.0 milliliters at a depth of upto about 4.5 milliliters.
 36. A culture container of a size to fit on aconventional microscope viewing stand for the identification of fungalpathogens comprising:(a) at least three well means for containing fungaldifferentiation media, the dimensions of each of said three well meansadapted to contain a quantity of up to about 5.0 milliliters of media ata depth of up to about 4.5 millimeters, each of said well means for thegrowth of a specimen; and (b) a first fungal differentiation mediapresent in one of said well means in the amount of said quantitycomprising purified saponin, oxgall, a substrate for phenol oxidase,ammonium ions, a carrying agent, and water, a second fungaldifferentiation media present in another of said well means in theamount of said quantity comprising a media effective to induce adifferent observable effect in at least one species of fungi subjectedto a previous carbohydrate depletion step as compared to another speciesof fungi subjected to said carbohydrate depletion step, wherein saidsecond fungal differentiation media is adapted in compositin to producean observable effect of positive or negative carbohydrate assimilationin carbohydrate depleted fungal cells within from about 3 to about 24hours from inoculation, and a third fungal differentiation media in thethird of said well means adapted in composition to induce a differentobservable effect in a fungal species metabolizing urea as compared to afungal species which does ot within from about 1 hour to about 3 hoursafter inoculation.
 37. The culture container of claim 36, furthercomprising a lid adapted to removably engage with said container. 38.The culture container of claim 36, wherein said quantity is from about2.5 milliliters to about 5.10 milliliters at a depth of up to about 4.5millimeters.